The growing emergency of multi-drug resistant-bacteria is a global concern, mostly in those countries where antibiotics are widely used in clinics. A number of pathogens like Staphylococcus aureus, Mycobacterium tuberculosis, some enterococci, Pseudomonas aeruginosa and many other Gram-negative bacteria have developed resistance against most traditional antibiotics as well as against those of new generation (Wenzel and Edmond 2000). It has therefore become increasingly important to develop new antibiotics. This demand urges the community of researchers and the pharmaceutical companies to consider new antimicrobial agents. Antimicrobial peptides are considered one of the best alternative to traditional antibiotics which generally cause the selection of resistant bacteria (Hancock and Sahl 2006). Most antibacterial peptides are components of the innate immunity of animals, including humans, plants and fungi (Zasloff, 2002). They usually consist of 6-50 amino acid residues and have a positive net charge. Cationic peptides interact selectively with anionic bacterial membranes and with other negatively charged structures such as LPS and DNA. Eukaryotic membranes, in their external layer, are normally less negatively charged than bacteria's, and, differently from bacterial membrane, they are also stabilized by cholesterol molecules. These differences are the basis of cationic peptides' specificity. The mechanism of action of cationic antimicrobial peptides is consequently due to their specific binding to bacterial membranes, which provokes cell permeation and, in some cases, metabolic pathways inhibition.
Many studies then, aimed to the identification and characterization of antimicrobial peptide sequences by studying their mechanism of action, their toxicity for eukaryotic cells and their therapeutic efficacy when administered topically or systemically. Unfortunately, two main problems hindered the development of antimicrobial peptide drugs so far. The first is that selectivity of natural antimicrobial peptides for bacteria is generally too low and they appear to be very toxic for eukaryotic cells, particularly erythrocytes, generating a high level of haemolysis. The second is linked to the generally short half-life of peptides in vivo. These are the main reasons for which only few cationic peptides reached the market in the last 10 years (polymyxin and daptomycin are two successful examples).
A few years ago, researchers began to concentrate on the identification of novel peptide sequences of non-natural origin, selected in the laboratory by rational design or screening of combinatorial libraries. The aim was to find peptides with better biological properties in terms of general toxicity and specificity for bacteria and improved half-life for drug development.
In the inventors' laboratory, a non-natural peptide sequence was identified, which showed a strong antimicrobial activity especially against Gram-negative bacteria (Pini et al, 2005). The peptide, (QKKIRVRLSA, SEQ ID No. 5, called M6) was obtained by rational modifications of a sequence identified from a combinatorial library, was synthesized in the MAP tetra-branched form where four identical peptide sequences are linked together by a lysine core. This molecule showed a high resistance to proteases and peptidases therefore overcoming the problem of short half-life (Bracci et al., 2003; Falciani et al., 2007). The branched antimicrobial peptide M6 has already been characterized for its biological activity against a number of bacteria, including several multi drug resistant clinical isolates, for its interactions with DNA, for its in vitro toxicity against several eukaryotic cell lines, as well as for its haemolytic activity, for its immunogenicity, for its in vivo toxicity when injected intraperitoneally or intravenously (Pini et al, 2007).
During all experiments carried out for M6 characterization we noted that different synthesis of M6 produced peptides with non homogeneus activities (batch to batch dissimilarity) (FIG. 1A). Mass spectrometry analysis revealed that the first aminoacid of M6, namely Gln, converts to pyroglutamic acid (FIG. 1B). The presence of the different peptide containing pyroglutramic acid and not Gln changes from batch to batch in an unpredictable percentage. The complete elimination of this secondary product is in facts impossible, firstly, because it is not easily discarded during HPLC purification because of the similar retention time with the main product, and secondly because it is continuously produced from the parent peptide when in solution. Since the pyroGlu-analogue showed a sensibly reduced antimicrobial activity with respect to the Gln analogue, the overall activity of the mixture varied from batch to batch (FIG. 1A).
In order to minimize batch to batch dissimilarity, in the perspective of a large scale peptide production for preclinical experiments and, possibly, for industrial manufacturing, we eliminated the first Gln residue from M6 and also replaced the first two Lys with Arg or alternated the first two residues with Lys and Arg. This produced the following 9-mer sequences deprived of the first Gln present in M6 sequence: KKIRVRLSA, SEQ ID NO. 1, called M33; RRIRVRLSA, SEQ ID NO. 2, called M34; KRIRVRLSA, SEQ ID NO. 3, called M35; RKIRVRLSA, SEQ ID NO. 4, called M36. These sequences are the object of the present application.
The new peptides were subjected to several characterizations described in the examples below.
Practically, the elimination of a single amino acid residue at the N-terminal end of M6 peptide sequence, and the possible alternation of Lys and Arg in the first two positions, produces a better antimicrobial activity, and does not cause any different behaviour in terms of side-toxicity and mechanism of action. Indeed, it produces a strong improvement in the synthesis liability, rendering the sequences of peptides M33, M34, M35 and M36 much more suitable for an industrial development with respect to M6. M33, M34, M35 and M36 sequences, thanks to the improved stability and batch to batch homogeneity, are ideal candidates for the development of antimicrobial drugs.